Citations

"Snapshot" images of localized Ca2+ influx into patch-clamped chromaffin cells were captured by using a recently developed pulsed-laser imaging system. Transient opening of voltage-sensitive Ca2+ channels gave rise to localized elevations of Ca2+ that had the appearance of either "hotspots" or partial rings found immediately beneath the plasma membrane. When the Ca2+ imaging technique was employed in conjunction with flame-etched carbon-fiber electrodes to spatially map the release sites of catecholamines, it was observed that the sites of Ca2+ entry and catecholamine release were colocalized. These results provide functional support for the idea that secretion occurs from "active zone"-like structures in neuroendocrine cells.

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.

Background The triceps tendon has been described as a single unit with contribution from each of the 3 heads of the muscle. An observation at the time of surgical repair of a triceps tendon injury led to an anatomical study to further define the anatomy of this tendon as it inserts on the olecranon. Hypothesis The medial head of the triceps has a tendon that is distinct from, and deep to, the common tendon of the long and lateral heads. Study Design Descriptive laboratory study and case report. Methods Eight cadaveric elbows were dissected to examine the triceps tendon. None of the specimens had any evidence of prior injury or surgery to the elbow. All specimens were fresh-frozen and stored at–4°C until they were thawed for use. Skin and subcutaneous tissue were removed, and the tendon of each head of the triceps was explored from the muscle to its bony insertion. Results In all 8 specimens, on gross inspection, the medial head had a separate insertion deep to the common insertion of the lateral and long heads. The muscle of the medial head extended further distally than did the long and lateral heads. The medial head was muscular to its deep insertion, with a small amount of tendon blended with the muscle distally. Histologic analysis demonstrated that the tendon of the medial head and that of the other 2 heads are confluent distally at their olecranon insertion. Conclusion The medial head of the triceps has a tendon that is distinct from, and deep to, the common tendon of the long and lateral heads on gross inspection. Histologic studies show the insertion of these 2 tendons is confluent. Clinical Relevance This anatomy has important implications for surgical repair of these tendon injuries. Rupture of the deep triceps insertion alone can occur and lead to weakness of elbow extension with the elbow flexed beyond 90°. Triceps strength should be tested with the elbow fully flexed when injury to the tendinous insertion is suspected.

Binding of human [ 125 I]luteinizing hormone to homogenates of luteinized rat ovaries is dependent upon time, temperature, and pH and is saturable. Injection of human chorionic gonadotropin in vivo or addition of unlabeled human chorionic gonadotropin or human or ovine luteinizing hormone in vitro inhibits binding, whereas follicle stimulating hormone or prolactin is without effect. The dissociation constant for binding of luteinizing hormone receptor is 0.79 nM. The number of binding sites is 94 femtomol/mg of wet weight. NaCl, KCl, MgSO 4 , and CaCl 2 at concentrations below 10 mM have no effect on binding, while all salts significantly inhibit binding at 150 mM. Binding of [ 125 I]luteinizing hormone to its receptors is destroyed by proteolytic enzymes and by phospholipase C and D. The total binding activity is quantitatively recovered in the 2000 × g pellet of the homogenate.

Abstract Context: We have recently introduced liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 25-hydroxyvitamin D2 (25OHD2) and 25OHD3 testing. During subsequent clinical use, we identified significantly elevated results in some infants. We hypothesized this might represent assay interference caused by C-3 epimers of 25OHD2 or 25OHD3. Objective: Our aims were to 1) determine the prevalence of C-3 epimers of 25OHD2 or 25OHD3 in human serum, and 2) identify the patient populations that might be affected. Study Design: We modified our LC-MS/MS method to allow detection of C-3 epimers. We retested specimens from four patient groups with the new method and an extracted RIA: 1) children less than 1 yr old, 2) children 1–18 yr old, 3) adults aged 20–87 yr with liver disease, and 4) adults aged 19–91 yr without liver disease. Results: In 172 children from group 1 with detectable 25OHD2 or 25OHD3, we identified C-3 epimers in 39 (22.7%). The epimers contributed 8.7–61.1% of the total 25-OHD. There was an inverse relationship between patient age and epimer percentage (r = 0.48; P < 0.002). The RIA gave accurate 25-OHD results that correlated with the modified LC-MS/MS method. No C-3 epimers were detected in any of the other groups. Conclusions: Significant concentrations of C-3 epimers of 25OHD2 or 25OHD3 are commonly found in infants. This can lead to overestimation of 25-OHD levels. Measurements in children less than 1 yr should therefore be performed with an assay that allows accurate detection of 25-OHD in the presence of its C-3 epimers.

Background— Vasoreactivity tests are fundamental in evaluating pulmonary arterial hypertension (PAH). Mutations of the transforming growth factor-β type II receptor gene, BMPR2 , predispose to the development of pulmonary hypertension and may alter the response to vasodilators. Previous investigations have not examined the relationship of BMPR2 mutations to vasoreactivity. Methods and Results— We identified 133 consecutive unrelated patients with either idiopathic or familial PAH. Sixty-six patients were excluded because we lacked either DNA samples (n=18) or complete data from a vasoreactivity test (n=48). The remaining 67 patients were screened for BMPR2 DNA sequence variations, and specific variations were confirmed by gene sequencing. The vasoreactivity of patients with nonsynonymous BMPR2 variations was compared with that of patients without nonsynonymous BMPR2 variations. We found nonsynonymous BMPR2 variations in 27 of 67 patients with idiopathic (n=16 of 52) or familial (n=11 of 15) PAH. Vasoreactivity was identified in 3.7% of 27 patients with nonsynonymous BMPR2 variations and in 35% of 40 patients without nonsynonymous BMPR2 variations ( P =0.003). Five of the 27 nonsynonymous variations occur commonly in healthy individuals. None of the remaining 22 patients with BMPR2 variations demonstrated vasoreactivity, and the analysis remained unchanged when we assumed that nonsynonymous BMPR2 variations were present in all 15 patients with familial PAH. Conclusions— Patients with familial or idiopathic PAH and nonsynonymous BMPR2 variations are unlikely to demonstrate vasoreactivity. Further trials are required to determine whether long-term therapy can be directed by tests for BMPR2 variations.

Background— The importance of completeness of revascularization by percutaneous coronary intervention in patients with multivessel disease is unclear in that there is little information on the impact of incomplete revascularization outside of randomized trials. The objective of this study is to compare long-term mortality and subsequent revascularization for percutaneous coronary intervention patients receiving stents who were completely revascularized (CR) with those who were incompletely revascularized (IR). Methods and Results— Patients from New York State’s Percutaneous Coronary Interventions Reporting System were subdivided into patients who were CR and IR. Then subsets of IR patients were contrasted with CR patients. Differences in long-term survival and subsequent revascularization for CR and IR patients were compared after adjustment for differences in preprocedural risk. A total of 68.9% of all stent patients with multivessel disease who were studied were IR, and 30.1% of all patients had total occlusions and/or ≥2 IR vessels. At baseline, the following patients were at higher risk: those who were older and those with more comorbid conditions, worse ejection fraction, and more renal disease and stroke. After adjustment for these baseline differences, IR patients were significantly more likely to die at any time (adjusted hazard ratio=1.15; 95% confidence interval, 1.01 to 1.30) than CR patients. IR patients with total occlusions and a total of ≥2 IR vessels were at the highest risk compared with CR patients (hazard ratio=1.36; 95% confidence interval, 1.12 to 1.66). Conclusions— IR with stenting is associated with an adverse impact on long-term mortality, and consideration should be given to either achieving CR, opting for surgery, or monitoring percutaneous coronary intervention patients with IR more closely after discharge.

AbstractAlthough ultrasonic and computed tomographic technology is continuing to evolve, these already sophisticated imaging tests have become the primary methods of evaluating the patients with aortic aneurysms. Both modalities are accurate and noninvasive. The major value of these techniques to the surgeon is in assessing those patients in whom the diagnosis is in doubt and in following patients in whom it has been elected to observe a small stable aneurysm. Changes in an aneurysm can be detected on serial examinations which might indicate the need for surgery in spite of adverse patient risk factors, and these techniques may provide a means of detecting false aneurysms in patients who are difficult to assess accurately by physical examination. Ultrasound and computed tomography are very helpful techniques in the diagnosis and assessment of aortic aneurysms. These diagnostic modalities are not required in every patient and should be used discriminately and when indicated.